Method for stimulating a host immune system

ABSTRACT

A method of manipulating allogeneic cells for use in allogeneic cell therapy protocols is described. The method provides a composition of highly activated allogeneic T-cells which are infused into immunocompetent cancer patients to elicit a novel anti-tumor immune mechanism called the “Mirror Effect”. In contrast to current allogeneic cell therapy protocols where T-cells in the graft mediate the beneficial graft vs. tumor (GVT) and detrimental graft vs. host (GVH) effects, the allogeneic cells of the present invention stimulate host T-cells to mediate the “mirror” of these effects. The mirror of the GVT effect is the host vs. tumor (HVT) effect. The “mirror” of the GVH effect is the host vs. graft (HVG) effect. The effectiveness and widespread application of the anti-tumor GVT effect is limited by the severe toxicity of the GVH effect. In the present invention, the anti-tumor HVT effect occurs in conjunction with a non-toxic HVG rejection effect. The highly activated allogeneic cells of the invention can be used in methods to stimulate host immunity. The method includes a complete HLA mis-matched setting in patients that have not had a prior bone marrow transplant or received chemotherapy and/or radiation conditioning regimens.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of and claims priority of U.S.patent application Ser. No. 13/099,867, filed May 3, 2011, now U.S. Pat.No. 8,728,534, which is a divisional of and claims priority of U.S.patent application Ser. No. 12/172,594, filed Jul. 14, 2008, now U.S.Pat. No. 7,943,180, which is a divisional of Ser. No. 10/838,454, filedMay 4, 2004, now U.S. Pat. No. 7,435,592, which is based on and claimsthe benefit of U.S. provisional patent application Ser. No. 60/470,171,filed May 13, 2003, U.S. provisional application Ser. No. 60/545,450,filed Feb. 18, 2004, U.S. provisional application Ser. No. 60/547,966,filed Feb. 26, 2004 and U.S. provisional application Ser. No.60/549,032, filed Mar. 1, 2004, the content of which is herebyincorporated by reference in its entirety.

FIELD OF INVENTION

The invention relates to a method for stimulating a host immune system.

BACKGROUND OF THE INVENTION

Allogeneic cell therapy is an important curative therapy for severaltypes of malignancies and viral diseases. Allogeneic cell therapyinvolves the infusion or transplant of cells to a patient, whereby theinfused or transplanted cells are derived from a donor other than thepatient. Types of allogeneic donors that have been utilized forallogeneic cell therapy protocols include: HLA-matched siblings, matchedunrelated donors, partially matched family member donors, relatedumbilical cord blood donors, and unrelated umbilical cord blood donors.The allogeneic donor cells are usually obtained by bone marrow harvest,collection of peripheral blood or collection of placental cord blood atbirth. This requirement for a matched donor is a major limitation ofallogeneic cell therapy protocols. It is an object of this invention toprovide a method of allogeneic cell therapy that is effective withoutthe requirement for HLA matching.

Allogeneic cell therapy methods have been practiced in the bone marrowtransplant (BMT) setting for over 30 years (Kai and Hara 2003). Thesemethods involve treatment of patients with high dose (myeloablative)chemotherapy and/or radiation. This myeloablative conditioning resultsin destruction of the bone marrow leading to the loss of a functioningimmune system. Thus, these patients must be “rescued” by allogeneic celltransplant to replace the destroyed bone marrow and restore immunity.

The ability of myeloablative conditioning followed by allogeneic BMT orstem cell transplantation (SCT) to cure certain hematologicalmalignancies is widely recognized. The anti-tumor effect mediated by theallogeneic cell transplant is known as the graft vs. tumor (GVT) effect(also called the graft vs. leukemia effect and the graft vs. malignancyeffect and the graft vs. myeloma effect). GVT activity after allogeneiccell therapy is known to be effective in treating several cancers,including myeloid leukemias (Gale and Champlin 1984), lymphoid leukemias(Rondon, Giralt et al. 1996, multiple myeloma {Tricot, 1996 #2730) andbreast cancer (Eibl, Schwaighofer et al. 1996).

However, allogeneic BMT has a treatment related mortality of 30-35%(Frassoni, Labopin et al. 1996). The high risk of transplant relatedmortality has limited the use of this treatment mostly to otherwisehealthy patients with hematological malignancies. It is an object ofthis invention to significantly reduce or eliminate the treatmentrelated mortality of allogeneic cell therapy in order to make thetreatment available to a broader spectrum of patients and diseaseindications.

The GVT effect was discovered when it was observed that relapse rateswere significantly lower in patients that received an allogeneic BMTcompared to patients that received an autologous BMT. This led to thediscovery that the reduced relapse rate was mediated by anti-tumorreactions of lymphocytes contained in the allograft (GVT effect)(Weiden, Sullivan et al. 1981).

Direct evidence of the power of the GVT effect was first provided whenpatients with chronic myelogenous leukemia (CML) who relapsed afterallogeneic BMT were put in complete remission after an infusion ofallogeneic lymphocytes (a procedure known as Donor Lymphocyte Infusionor DLI). DLI treatment has since been shown to frequently cause completeremissions in relapsed cancer patients following allogeneic BMT, despitecomplete resistance of such tumor cells to maximally tolerated doses ofchemotherapy/radiation (Slavin, Naparstek et al. 1995; Slavin, Naparsteket al. 1996; Slavin, Naparstek et al. 1996) (See also Slavin U.S. Pat.Nos. 5,843,435 and 6,143,292).

The observation that DLI treatment alone, without chemotherapy, couldhave an anti-tumor effect has led to a paradigm shift in the treatmentof malignancy. A new generation of therapies has emerged where the focusis on the GVT effect, rather than the cytotoxic effect ofchemotherapy/radiation. This new generation of allogeneic cell therapyprotocols is known as a “Mini-Transplant” (for example, see U.S. Pat.No. 6,544,787 issued to Slavin and U.S. Pat. No. 6,558,662 issued toSykes, et al.).

Mini-Transplant procedures involve a first round of low dose,non-myeloablative chemotherapy conditioning of a patient. The low dosechemotherapy conditioning is not provided for the purpose of tumorreduction, but rather is designed to only weaken the immune systemenough to prevent rejection of an allogeneic donor cell infusion.Conditioned patients are infused with non-manipulated allogeneiclymphocytes or stem cells which engraft in the patients and subsequentlymediate a GVT effect.

Patients with successfully engrafted allogeneic cells develop immunesystems which are partially of self origin and partially of theallogeneic graft origin. Patients in this immunological state are knownas “chimeras”. The conditioning regimen enabling chimera formationusually includes administration of one or more chemotherapy conditioningagents, such as purine analogs like fiudarabine, alkylating agents suchas busulfan and/or cyclophosphamide, and/or anti-leukocyte globulins(see U.S. Pat. No. 6,544,787 issued to Slavin).

These Mini-Transplant protocols have proven to be very effective in thetreatment of hematological malignancies and are less toxic than the highdose myeloablative regimens (Champlin, Khouri et al. 1999; Champlin, vanBesien et al. 2000); (Grigg, Bardy et al. 1999); (Slavin, Nagler et al.2001; Slavin, Or et al. 2001). Mini-Transplants have also been shown tobe effective in chemotherapy resistant metastatic disease (Childs,Chernoff et al. 2000; Childs 2000; Childs and Barrett 2002; Childs2002).

While the GVT effect has been described as the most powerful andeffective anti-tumor mechanism ever observed in the treatment of humanmalignancies (van Besien, Thall et al. 1997) (Eibl, Schwaighofer et al.1996) (Deno, Rondon et al. 1998), the clinical application of GVT isstill severely limited due to the toxicity associated with allogeneiccell infusions. The major complication of allogeneic cell therapy is thecondition known as graft vs. host (GVH) disease. GVH disease occurs whendonor T-cells react against antigens on normal host cells causing targetorgan damage, which often leads to death. The principal target organs ofGVH disease are the immune system, skin, liver and intestine.

There is an urgent need to develop methods to separate the beneficialGVT effect from the detrimental GVH effect in allogeneic cell therapy.However, this has proven to be very difficult, as it appears that GVTand GVH are intimately related processes, with the same donor T-cellsresponsible for both effects. It is an object of this invention todescribe an allogeneic cell therapy method which provides an anti-tumoreffect without the toxicity associated with GVH disease.

GVH disease occurs secondary to mismatches between histocompatibilityantigens (HLA) between the donor and the recipient. Attempts to performallogeneic BMT between strongly HLA-mismatched donor-recipient pairshave been associated with a prohibitively high incidence of severe GVHdisease and failure of the allogeneic cell infusions to engraft.Therefore, allogeneic cell therapy normally requires matching of HLAantigens between donor and recipient. However despite matching of HLAidentity, substantial numbers of patients still develop GVH disease,presumably due to differences in minor HLA antigens.

The requirement for an HLA matched donor severely limits the applicationof allogeneic cell therapy. Only approximately one of every threepatients has an HLA-matched sibling or can find a phenotypically matchedunrelated donor, and therefore the majority of patients do not have theoption of allogeneic cell therapy. Furthermore, a large fraction ofcancers, including leukemias and lymphomas, afflict older patients whoare more prone to the development of GVH disease than are youngerpersons, and who therefore are not generally considered candidates forallogeneic cell therapy, despite the lack of other curative options. Inaddition, the immunosuppressive drugs used for GVH disease prophylaxisalso increase the risk of secondary infection and increase the relapserates for certain types of leukemia.

Accordingly, there is a great need to reduce or eliminate the toxicityassociated with GVH disease in allogeneic cell therapy protocols whilemaintaining or increasing the GVT effect in order that the therapy couldbe utilized to benefit a greater population of patients.

It is an object of this invention to describe an allogeneic cell therapymethod that elicits an anti-tumor effect at least as effective as theGVT effect without the associated GVH disease toxicity.

It is an additional object of this invention to describe an allogeneiccell therapy method with reduced treatment related toxicity byeliminating the requirement for a previous allogeneic BMT orchemotherapy conditioning regimen in order to benefit from the therapy.

It is an additional object of this invention to describe a method ofallogeneic cell therapy that does not require an HLA-matched donor.

SUMMARY OF THE INVENTION

The invention disclosed herein relates to a product comprised ofallogeneic cells of which at least a portion are T-cells, whereby theallogeneic T-cells are expanded and differentiated ex-vivo, and are usedas an allogeneic cell therapy for the stimulation of the host immunesystem in humans without GVH toxicity, and whereby said allogeneic cellsare subsequently rejected by the host immune system.

The invention disclosed herein also relates to a product described abovewhereby the allogeneic cells are chosen without regard for HLA-matchwith the recipient, or to allow for the maximum mismatch of HLAhaplotype with the intended patient population, thereby ensuring themaximum allogeneic potential and subsequent host immune response to theproduct.

The invention disclosed herein also relates to a product described abovewhereby the allogeneic cells are capable of stimulating an effectivehost immune response against a tumor when infused into patients thathave not received a prior allogeneic BMT.

The invention disclosed herein also relates to a product described abovewhereby the allogeneic cells are capable of stimulating an effectivehost immune response against a tumor when infused into a patient thathas not been subjected to immunosuppressive conditioning regimens.

The invention disclosed herein also relates to a product described abovewhereby the allogeneic cell therapy stimulates an immune response inpatients by stimulating the production of inflammatory “Type 1”monokines and lymphokines in the host.

The invention disclosed herein also relates to a product described abovewhereby the allogeneic cell therapy stimulates an immune response inpatients by activating components of host innate and/or Th1 adaptiveimmunity.

The invention disclosed herein also relates to a product described abovewhereby the allogeneic cell therapy stimulates the production ofcytokines which enhance the immunogenicity of tumors.

The invention disclosed herein also relates to a product described abovewhereby the allogeneic cells directly kill tumors so as to cause thetumor associated antigens to be available for stimulating host Type 1adaptive immunity.

The invention disclosed herein also relates to a method of producing aproduct as described above, whereby the allogeneic T-cells contained inthe product are in a state of enhanced activation.

The invention disclosed herein also relates to a method for stimulatinga host immune system by collecting the mononuclear cells from anunrelated donor, activating T-cells within the mononuclear cellpopulation, and administering the activated T-cells to a host having ahost immune system whereby the activated T-cells are rejected by thehost immune system while stimulating the host immune system to mediatean effective immune response against a resident disease. The host mayhave a resident disease such as hematological malignancy, a solid tumor,a solid tumor that has metastasized or a viral infection. The donor isselected without regard to histocompatibility to the host, and maximumhistocompatibility mismatch is preferred. The host also preferablyshould not have had a prior bone marrow transplant and should notpreferably have received any immunosuppressive chemotherapy and/orradiation conditioning regimens designed to allow engraftment of theallogeneic donor cell infusions.

The method further includes that the T-cells are preferably CD4+ T-cellsand that a majority of the CD4+ T-cells differentiate after ex-vivoactivation from CD45RA+, CD62L^(hi) naïve cells into CD45RO+, CD62L^(lo)memory cells, and wherein such cells produce Type 1 cytokines such asIL-2, IFN-gamma, TNF-alpha and do not produce Type 2 cytokines such asIL-4, IL-10 and TGF-beta.

The invention disclosed herein also includes such CD4+ T-cells whichafter ex-vivo activation express CD40L and/or TRAIL on the cell surface.

Preferably, the T-cells are activated by cross-linking of anti-CD3 andanti-CD28 mAbs applied to the cell surface of the T-cells. Preferablyanti-CD3 and anti-CD28 mAbs applied to the surface of said T-cells arecross-linked by association with biodegradable microspheres coated withan agent reactive against said mAbs.

The invention disclosed herein also includes wherein greater than 90% ofthe T-cells are in a state of activation just prior to and at the timeof contacting the host immune system, and in the preferred embodimentgreater than 95% of the T-cells are activated at the time ofadministration to the host and just prior to contacting the host.

The method also includes wherein T-cells are continuously exposed to anactivating stimulus for at least six days prior to infusion in the host.T-cells are preferably activated while being maintained at celldensities of at least 10⁷ cells/ml to maximize cell to cell contact.Such cell to cell contact serves to enhance the state of activation ofthe allogeneic T-cells.

In another embodiment, the method includes wherein the T-cells areadministered with anti-CD3 and anti-CD28 mAbs applied to the surface ofthe allogeneic T-cells and wherein the mAbs are cross-linked byassociation with and inclusion of biodegradable micro spheres coatedwith an agent reactive against the mAbs.

The method also includes wherein T-cell administration stimulatesproduction of Type 1 cytokines, and such cytokines include at least oneof the following: IL-1, IL-2, IL-12, IL-15, IFN-gamma, IFN-alpha,IFN-beta, TNF-alpha, and TNF-beta. Such cytokines stimulate immunityincluding host innate immune function. The method also includes whereinthe activated T-cell administration activates host dendritic and/ormacrophage cells.

The invention also includes wherein the activated allogeneic T-celladministration and subsequent rejection of the activated T-cellsstimulates an immune response against a host resident disease.

The invention also includes a method wherein the ex-vivo activatedallogeneic T-cells are cryopreserved prior to formulation andadministration to the host.

The invention also includes a composition of allogeneic T-cells labeledwith anti-CD3 and anti-CD28 mAbs cross-linked with biodegradablemicrospheres coated with an agent reactive against said mAbs. Thelabeled allogeneic T-cells and associated biodegradable microspheres aresuspended in a media suitable for intravenous infusion. Such T-cells andassociated biodegradable microspheres are suspended at a cell density of10⁷ cells/ml or greater, and preferably in a flexible container or in asyringe. The T-cells labeled with anti-CD3 and anti-CD28 may also becryopreserved prior to formulation and administration.

The present invention also includes an allogeneic cell material thatelicits a host vs. tumor (HVT) and host vs. graft (HVG) response whencontacted with a tumor-bearing host immune system without eliciting atoxic graft vs. host (GVH) response. The allogeneic cell materialcontains ex-vivo activated T-cells and wherein said activated T-cellsare preferably CD4+ T-cells.

The present invention also includes an allogeneic cell material thatcauses apoptosis of tumors when administered to a tumor-bearing host.The allogeneic cell material contains activated allogeneic T-cells, andsuch T-cells are preferably CD4+ cells. Such CD4+ cells should expressFasL and/or TRAIL on the cell surface, preferably at high density. Suchactivated T-cells preferably differentiate into memory cells expressingCD45RO and CD63L^(lo) after ex-vivo activation. Such allogeneic T-cellsshould express one or more of the following cytokines: IL-2, IL-15,IFN-gamma, and TNF-alpha and express surface FasL and/or TRAIL uponadministration to the host.

The present invention also includes a composition comprising a treatmenteffective amount of a population of allogeneic cells, of which at leasta portion are T-cells, and whereby said T-cells are labeled with anactivating effective amount of one or more monoclonal antibodies, orportions thereof, and a cross-linking effective amount of an agentreactive against the monoclonal antibodies. T-cells of such compositionare preferably labeled with anti-CD3 and anti-CD28 mAbs. The agentreactive against the mAbs is preferably coated on biodegradablemicrospheres. The allogeneic T-cells and associated biodegradablemicrospheres are suspended in a media suitable for intravenous infusion.Such labeled T-cells and associated cross-linking biodegradablemicrospheres are suspended at a cell density of 10⁷ cells/ml or greaterin a flexible container or in a syringe. The composition may becryopreserved prior to infusion.

In preferred embodiments, the allogeneic cells used in the presentinvention are purified T-cells which have been activated ex-vivo,preferably CD4+ T-cells, more preferably CD4+ T-cells that havedifferentiated into effector or memory cells and produce high levels ofType 1 cytokines, such as IL-2, IL-15, IFN-gamma, TNF-alpha and alsoexpress, preferably at high density, effector molecules such as CD40L,TRAIL and FasL on the cell surface.

In another preferred embodiment, the allogeneic T-cells for infusion areprocessed ex-vivo by a method which maintains the cells at high celldensity (10⁷ cells/ml or greater) in continuous contact with T-cellactivating agents.

In another preferred embodiment, the allogeneic T-cells for infusion areformulated in media suitable for infusion containing activating agentsas a means to maintain the activation state of the T-cells from harvestthrough infusion.

In another preferred embodiment, greater than 90%, or preferably greaterthan 95% of the infused allogeneic T-cells continue in a state ofenhanced activation at the time of infusion into the patient.

The “Mirror Effect”

In the prior art allogeneic cell therapy protocols, T-cells in the graftare responsible for mediating the beneficial GVT effect and thedetrimental GVH effect of the therapy. In order to accomplish theobjectives of this invention, a new mechanism is described whereby theT-cells in the graft do not directly mediate the immune effects, butinstead act to stimulate the host immune system to mediate an effectiveimmune response against a resident disease.

The host immune response elicited by the method of this invention is the“mirror” of the GVT/GVH effects of prior art allogeneic cell therapyprotocols. The “mirror” of the normally observed GVT effect inallogeneic cell therapy is the host vs. tumor (HVT) effect. The “mirror”of the normally observed GVH effect in allogeneic cell therapy is thehost vs. graft (HVG) effect. The HVT/HVG effects are hereinaftercollectively called the “Mirror Effect”.

Unlike the extremely toxic GVH component of prior art allogeneic celltherapy protocols, the HVG component of the Minor Effect results only inthe non-toxic rejection of the graft cells. Thus in the presentinvention, the HVT anti-tumor component of the Mirror Effect occurswithout the toxicity of GVH. It is understood in the art that aneffective anti-tumor immune response can also be effective against avariety of pathogens, including viruses.

In the present invention, the rejection of the graft (HVG) is a desiredcomponent of the Mirror Effect. Therefore, it is not necessary to treatthe recipient patients with immunosuppressive conditioning regimens inorder to prevent rejection of the graft, as is required in prior artallogeneic cell therapy protocols. In addition, unlike the GVH componentof prior art allogeneic cell therapies, the HVG component of the MirrorEffect is a non-toxic immunological event. In prior art allogeneic celltherapy protocols it is necessary to select HLA-matched donors in orderto reduce the toxic effects of the GVH effect. Since the HVG componentof the Mirror Effect is non-toxic, it is not necessary to use anHLA-matched donor in the present invention as a means to limit theeffect. In fact, it is preferable in the practice of the presentinvention to use allogeneic donors that have complete HLA disparity withthe host. The greater the HLA disparity, the stronger the stimulation ofthe host immune response.

In prior art allogeneic cell therapy protocols the beneficial GVT effectand the detrimental GVH effect are intimately and proportionallyrelated. There are at least two forces which serve to limit themagnitude of the GVT effect in these prior art allogeneic cell therapyprotocols that are not present to limit the anti-tumor HVT component ofthe Mirror Effect. These factors are: (1) the development of hosttolerance to the donor cells enabling engraftment and chimerism; and (2)GVH prophylaxis with immune suppressive drugs.

Host tolerance is an immune mechanism where the host immune systemceases to respond against the graft cells and the graft cells cease torespond against the host cells. The mechanism of tolerance is correlatedwith immunosuppressive mechanisms resulting in the undesired reductionof the GVT effect. GVH prophylaxis is required in prior art allogeneiccell therapy protocols in order to limit the extent of GVH disease.Because of the proportional relationship between GVT and GVH, thelimitation of the GVH component by prophylaxis proportionally limits theGVT component.

In the Mirror Effect, the HVT and HVG components are also intimately andproportionally related. The HVT component provides a more powerfulanti-tumor effect than the GVT effect. This is because the HVG effectdoes not require immunosuppressive treatment to limit the extent of therejection response. In this way, unlike the GVH effect, the HVG effectcan be allowed to reach its natural conclusion (complete rejection). Theproportional nature of the HVG effect with the anti-tumor HVT effectresults in a more powerful anti-tumor component occurring concurrentlyand in proportion to the rejection response. It is preferred, therefore,that the rejection response be enhanced, rather than limited, by the useof completely mis-matched allogeneic cells. The enhanced HVT anti-tumoreffect also occurs because, unlike the GVT effect, the HVT effect doesnot occur in the limiting environment of tolerance induction. In thepresent invention, rather than an immunosuppressive tolerance effect, apowerful Th1-mediated allo-rejection response is mediated. This Th1allo-rejection response has a by-stander effect which helps to sustainand amplify the HVT effect.

Accordingly, the allogeneic cell therapy of the present inventionprovides significant advantages for the treatment of malignancies overprior art methods: (1) enhanced anti-tumor effect (HVT) compared to theGVT effect; (2) potentially curative anti-tumor effect without lethaltoxicity; (3) elimination of the requirement for a matched donor; (4)elimination of the need for immunosuppressive conditioning prior totherapy; and (5) elimination of the need for a prior allogeneic BMT (asis required in DLI protocols).

Ex-Vivo Manipulation Requirement

In order for a population of allogeneic cells to induce the MirrorEffect, the population must contain T-cells. To be effective, theallogeneic T-cells must be manipulated ex-vivo in a manner that causesactivation. The T-cells need not be activated with specific antigens,but preferably are activated polyclonally. Preferably the activatedT-cells proliferate at least 4 generations and differentiation to obtaineffector function. T-cells that express effector function produce Type 1cytokines including IL-2, IFN-gamma and TNF-alpha, express activationmarkers such as CD25 and HLA-DR, and express effector molecules such asTNF superfamily molecules such as TRAIL, LIGHT, CD40L and FasL. Inanother preferred embodiment, the ex-vivo activated allogeneic T-cellsfurther differentiate into memory cells that express CD45RO andCD62L^(lo).

T-cells generally require two signals to be activated. The first signalrequired for activation occurs by stimulation of the T-cell antigenreceptor (TCR), a multisubunit immune recognition receptor thatassociates with the CD3 complex and binds to peptides presented by themajor histocompatibility complex (MHC) class I (CD8+ T-cells) and classII (CD4+ T-cells) proteins on the surface of antigen-presenting cells(APCs). The first signal can be provided by immobilized anti-CD3 mAb.The second signal is typically delivered through co-stimulatorymolecules. The major co-stimulatory signal occurs when a member of theB7 family ligands CD80 or CD86 on an activated antigen-presenting cell(APC) binds to CD28 on a T-cell. The second signal can be provided bysoluble or immobilized anti-CD28 mAb. For purposes herein, T-cells at acell density of less than 10⁶ cells per ml and activated with anti-CD3and anti-CD28 are termed “standard conditions”.

In the present invention, the T-cells should enter and maintain a stateof “enhanced activation” prior to infusion. For purposes of the presentinvention, “enhanced activation” shall mean a T-cell that has beenactivated in a manner that results in enhanced proliferationcharacteristics (i.e., proliferation greater than a population activatedunder standard conditions) and terminally differentiates to performenhanced effector functions, including enhanced cytokine production andenhanced expression of effector molecules when compared to T-cellsactivated under standard conditions.

In a preferred embodiment, a population of allogeneic T-cells withenhanced activation characteristics are produced by a process thatinvolves: (1) collection of mononuclear cells by leukapheresis; (2)purification of CD4+ cells from the mononuclear cells; (3) contactingthe CD4 cells with cross-linked anti-CD3 and anti-CD28 mAbs; (4)maintaining constant contact of the cross-linked CD3/CD28 mAbs on theCD4 cells for at least a period of 6 days; (5) maintaining over the sameminimum 6 day period enhanced cell to cell contact; and (6) formulatingand then infusing the CD4 cells at the peak of proliferation andcytokine production while maintaining constant contact with thecross-linked CD3/CD28 mAbs.

Prior art allogeneic cell therapies generally involve infusion ofnon-manipulated allogeneic cells into a host with an immune system thathas been suppressed by chemotherapy and/or radiation conditioningregimens. These prior art procedures result in engraftment of theallogeneic cells, which in turn mediate the GVT/GVH effects. Ex-vivomanipulation of the graft cells in this setting may increase GVTeffects, but also results in exacerbation of the toxic GVH effects.Infusion of non-manipulated allogeneic cells into a host with an intactimmune system results only in rejection of the allogeneic cells (HVG)without any anti-tumor effect. Therefore, the manipulation of theallogeneic graft cells is a required embodiment for eliciting the MirrorEffect.

The present invention is designed to take advantage of the knownmechanisms of the GVT effect in prior art allogeneic cell therapyprotocols, including the key role of the innate immune system ininitiating effective, appropriate and targeted adaptive immune responsesand the role Type 1 cytokines have in bridging innate and adaptiveimmunity. The method of the present invention is designed to mirror theknown mechanisms that mediate the GVT effect within the host (ratherthan within the graft).

Patients with tumors have immune responses that have failed to protectagainst the tumor. This can be for many reasons, including the initialimprinting of a Type 2 immune response against the tumor and/or due totumor immunoavoidance mechanisms. The GVT effect of prior art allogeneiccell therapy protocols is capable of overcoming these limitations insome settings. The key to overcoming these limitations is thestimulation of an inflammatory Type 1 cytokine storm (described in moredetail below) in the context of de novo shedding of tumor antigenresulting from the conditioning regimens, as well as the activation ofcomponents of innate immunity in the graft and the subsequent imprintingof a de novo graft-mediated Type 1 adaptive immune response against thetumor.

The present invention is designed to elicit these mechanisms in the hostrather than the graft. Accordingly, the allogeneic cells of the presentinvention are designed to elicit a Type 1 cytokine storm, de novoshedding of tumor antigen, activation of components of host innateimmunity leading to a host Type 1 adaptive immune response against thetumor.

The major cellular components of the innate immune system consists ofmacrophages, NK cells, neutrophils, gamma-delta T-cells, alpha-betaintermediate T-cells and NKT cells. The activation of the host innateimmune response to tumors results in the killing of tumors, shedding oftumor associated antigens (TAA) into draining lymph nodes, enhancedpresentation of TAA to naïve T-cells and also plays an instructive rolein emanating the subsequent Type 1 adaptive immune response.

Components of the adaptive immune response coordinate to specificallyeliminate the tumor. The adaptive immune response is characterized byits specificity for a tumor and the ability to distinguish between selfand non-self. The major cellular components of the adaptive anti-tumorimmune response consist of CD4+ T-cells and CD8+ T-cells. Antigenpresenting cells (APC), Such as activated dendritic cells, plasmacytoiddendritic cells and macrophages serve to bridge between the innate andadaptive immune compartments by presenting TAA to components of theadaptive immune system.

In order to elicit the full benefit of the Mirror Effect, the allogeneiccell infusion must be manipulated ex-vivo so that the cells are capableof eliciting a cascade of immunological mechanisms upon infusion. Thefirst mechanism the allogeneic cells should elicit is a Type 1 cytokinestorm consisting of both monokines and lymphokines. In the presence ofthis cytokine storm, the following mechanisms should also be elicited:(1) the activation of dendritic cells; (2) the shedding of TAA; and (3)development of a Type 1 adaptive immune response.

Type 1 Cytokine Storm

Two helper (CD4) T-cell subsets, Th1 and Th2, have been defined whichare characterized by distinct and mutually exclusive patterns ofcytokine production. Th1 cells produce IL-2, IFN-gamma and TNF-alpha andare responsible for inducing inflammatory, cell-mediated immuneresponses that are protective against tumors, intracellular bacteria andviral infections. Th2 cells produce IL-4 and IL-10 and enhance humoralimmune responses that are generally effective against certainextracellular bacterial and parasitic infections.

It has also been discovered that other types of immune cells exhibitdistinct cytokine polarity, including CD8 T-cells, NKT-cells, NK cellsand dendritic cells. A typical immune response will thus have complexmixtures of effector cells and cytokines. Cytokines are an importantcomponent of any immune response and the balance of cytokines inresponse to a tumor or pathogen is usually determinative of the type ofimmune response that will be generated. The type of immune responsegenerated is determinative of whether the tumor or pathogen will beeradicated or allowed to persist.

To assist in the categorization of immune responses, they can becharacterized as ‘Type 1’ or ‘Type 2’, depending on the dominantcytokine profile. Type 1 responses are dominated by inflammatorycytokines and Type 2 responses are dominated by cytokines which suppresscellular immunity. Cytokines have been categorized as beingcharacteristic of Type 1 or Type 2 immune responses. The cytokines aredefined functionally as Type 1 or Type 2, corresponding to their abilityto support cellular immunity and suppress humoral immunity (Type 1) orsupport humoral immunity and suppress cellular immunity (Type 2). Type 1cytokines include IL-2, IL-12, IL-15, IFN-gamma, IFN-alpha and IFN-beta.Type 2 cytokines include IL-4, IL-5, IL-6, IL-10, IL-13 and TGF-beta.(Belardelli and Ferrantini 2002). In the case of tumors, a Type 1 immuneresponse is critical for protective immunity (Nishimura, Nakui et al.2000).

A common mechanism for the prior art GVT and GVH effects is a “cytokinestorm” of Type 1 cytokines (Fowler, Breglio et al. 1996; Das, Imoto etal. 2001); (Carayol, Bourhis et al. 1997; Tanaka, Imamura et al. 1997;Blazar, Taylor et al. 1998; Flanagan, Jennings et al. 1999; Fowler andGress 2000). The Type 1 cytokine storm activates components of both theinnate and adaptive immune responses in the donor lymphocyte population(Antin and Ferrara 1992; Blazar, Korngold et al. 1997; Tanaka, Imamuraet al. 1997). In the present invention, the infusion of the allogeneiccells is designed to elicit the same Type 1 cytokine storm. However, thepresent invention is designed so that the cytokine storm activatescomponents of the innate and adaptive immune responses of the hostlymphocyte population, rather than of the donor lymphocyte population.In order to assure that only host components are activated, it ispreferred that the host is immunocompetent and that the allogeneic cellpopulation infused be devoid of innate immune cells and enhanced inT-cells, preferably enhanced in CD4+ T-cells.

In preferred embodiments, the allogeneic T-cells to be infused producehigh amounts of Type 1 cytokines, including IL-2, IL-15, TNF-alpha,TNF-beta, and IFN-gamma and do not produce IL-4, IL-10 or TGF-beta. Theinfused cells should be producing one or more of the Type 1 cytokines atthe time of infusion and while circulating in the patient blood. Toensure that the allogeneic T-cells are producing Type 1 cytokines at thetime of infusion and while circulating, the T-cells should be activatedwhen infused and maintain the activation status while in circulation.

To ensure that the T-cells are activated, they should be formulated withagents which deliver activation signals. For example, the T-cells canfirst be contacted with activating agents, such as mouse anti-human CD3and mouse anti-human CD28 mAbs. The mAbs on the surface of the T-cellscan then be cross-linked to deliver activation signals to the T-cells.In preferred embodiments, the cross-linking is accomplished by includingcoated biodegradable microspheres or nanospheres in the formulationmedia. The biodegradable spheres are coated with an agent which reactswith the activating agents on the T-cells and causes them to becross-linked and to deliver activation signals. Suitable coating agentsfor use with mouse mAbs include polyclonal anti-mouse antibodies ormonoclonal anti-Fc mAbs.

The method of the present invention comprises introducing a sufficientamount of activated allogeneic T-cells, preferably CD4+ T-cells, into ahost to stimulate host mononuclear cells to produce Type 1 cytokines,especially IL-1, IL-12, IL-15, IL-18, TNF-alpha, GM-CSF, IFN-alpha andIFN-gamma and not inducing significant production of IL-4, IL-10, IL-13and TGF-beta from host cells.

The production of the Type 1 cytokine storm is known to be important inthe link between the initial innate immune activation and the subsequentadaptive immune response in anti-tumor immunity (Belardelli andFerrantini 2002; Kadowaki and Liu 2002; Le Bon and Tough 2002).

Activation of Dendritic Cells

A key cell type in bridging innate immunity to adaptive immunity is thedendritic cell (DC). Therefore, it is important for eliciting the fulleffectiveness of the Mirror Effect for the allogeneic cell infusion toactivate host DC cells. The method of the present invention comprisesintroducing a sufficient amount of activated allogeneic T-cells,preferably CD4+ T-cells, into a host in order to stimulate theactivation and maturation of host DCs.

After activation, DCs are known to go through maturational stages inwhich they express cytokines and cell surface molecules critical for theinitiation and the control of innate and then adaptive immune responses(Langenkamp, Messi et al. 2000; Granucci, Vizzardelli et al. 2001). Inparticular, inflammatory Type 1 cytokines, such as TNF-alpha, macrophageinflammatory protein-1α (MIP-1α), IL-12 and SLAM, are stronglyupregulated after activation. IL-12 is produced by monocyte-derived DCsafter activation. Activated IL-12-producing DCs are then able to primeType 1 immune responses (Langenkamp, Messi et al. 2000).

Allogeneic T-cells prepared by the process of the present invention willexpress CD40L (CD154) on the cell surface. These CD40L expressingT-cells will activate host DCs. Ligation of CD40 on the DCs up-regulatescostimulatory/accessory molecule expression (MHC class II, CD58,CD80/CD86) that enhance antigen presentation by the DCs. Thisinteraction in turn is known to “prime” CD40L+ helper (CD4) andcytotoxic (CD8) T cells by up-regulating their IL-2 receptor expression,and is also known to lead to the expansion of both class II□ and classI□ dependent tumor-reactive T-cell pools.

Tumor Associated Antigen (TAA) Shedding

Shedding of TAA into the draining lymph nodes and the presentation ofthese antigens by activated DCs stimulates an adaptive immune response.TAA shedding is caused by the killing of tumor cells by immune effectorcells. In the present invention, tumor killing causing shedding of TAAis mediated by both direct and indirect mechanisms. The direct mechanismof tumor killing is mediated by the interaction of tumor cells withsurface molecules on the infused allogeneic cells and/or activated hostT-cells, such as FasL and TRAIL, which stimulate programmed cell death(apoptosis) of the tumor cells. The indirect mechanism includes theactivation of host innate immune effector cells such as NK cells andphagocytic macrophages.

The method of the present invention comprises introducing a sufficientamount of activated allogeneic T-cells, preferably CD4+ T-cells, into ahost in order to stimulate tumor lysis by elements of the innate immunesystem, such as NK cells. An additional method of the present inventioncomprises introducing a sufficient amount of activated allogeneicT-cells, preferably CD4+ T-cells, into a host which mediate tumorapoptosis through expression of TNFR effector molecules such as FasL andTRAIL. An additional method of the present invention comprisesintroducing a sufficient amount of activated allogeneic T-cells,preferably CD4+ T-cells, into a host which results in activation of hostT-cells, whereby such activated host T-cells express TNFR ligands suchas FasL and/or TRAIL and mediate apoptosis of tumor cells.

De-novo TAA shedding is an important component of the GVT effect, as itenables the reprogramming of the adaptive immune response from one thatallowed tumor growth (Type 2) to one that kills and protects against thetumor (Type 1). NK cells in the graft constitute major effector cells inthe GVT reaction (Voutsadakis 2003) that can mediate de-novo TAAshedding.

Similarly, activation of host NK cells is an important part of the HVTcomponent of the Mirror Effect. The Type 1 cytokine storm, resultingfrom the host response to the activated allogeneic T-cells of thepresent invention, is capable of activating host NK cells and stronglyupregulating their cytotoxic capacity.

Activated host NK cells have the ability to kill a wide variety of tumorcells spontaneously while sparing normal cells (Smyth, Hayakawa et al.2002). Importantly, NK cells recognize potential target cells withoutthe need for immunization or pre-activation compared with T cells, whichfirst require education by antigen-presenting cells. Furthermore, NKcells can recognize tumors that might evade T-cell killing by downregulation of MHC I molecules, a major tumor immunoavoidance mechanism.

Therefore, the method of the current invention causes the shedding ofTAA through the activation and upregulation of cytotoxic activity ofhost NK cells. Another mechanism for inducing the shedding of TAA by themethod of the current invention is to induce apoptosis in tumors. Onemechanism for the induction of apoptosis in tumors is by signalingthrough the death receptor called Fas (CD95). Binding of Fas with itsFas-ligand (FasL/CD154) induces programmed cell death (apoptosis).Another mechanism is by signaling through the TRAIL ligand by itscounter receptor TRAIL-R. Allogeneic T-cells produced by the method ofthe present invention express high levels of FasL and TRAIL. However,many tumors lose expression of Fas during the tumor progression processand many tumors also express FasL as a defense against apoptosis.Approximately 80% of tumor cell lines representing colon, lung, breast,skin, kidney and brain tumors are sensitive to TRAIL induced apoptosis.

The Type 1 cytokine storm elicited by the method of the presentinvention upregulates the expression of Fas on tumor cells, making themsusceptible to FasL-mediated apoptosis by the infused allogeneic T-cellsand susceptible to a fratricide type response to other tumor cellscaused by the upregulation of Fas on the FasL expressing tumor cells. Inaddition, the same cytokine storm activates host T-cells to express FasLwhich can then mediate the apoptosis of tumor cells. The same Type 1cytokine storm also upregulates MHC I, MHC II and co-stimulatorymolecules CD80 and CD86 on tumor cells, making the tumors susceptible toCTL-mediated killing in a subsequent adaptive immune response.

Type 1 Adaptive Immune Response

The method of the present invention induces the activation of host DCsand the de novo shedding of TAA in the context of a Type 1 cytokinestorm. These are the conditions required for the de novo development ofa host Type 1 adaptive immune response against a tumor.

The method of the present invention comprises introducing a sufficientamount of activated allogeneic T-cells, preferably CD4+ T-cells, into ahost in order to stimulate a host Type 1 adaptive immune responsedirected against the host tumor. This mechanism is analogous to a tumorvaccine, whereby the antigen is shed by in-situ tumor killing and theType 1 cytokine storm serves as an adjuvant for the development of thesubsequent adaptive immune response. The shedding of TAA in the host andthe vaccination effect of the present invention can be enhanced by theco-infusion of agents containing TAA, such as inactivated allogeneic orautologous tumors, specific TAA peptides, DNA coding TAA or cellsgenetically engineered to express TAA.

Some cancer patients develop a Type 1 adaptive immune response against atumor, which fails to protect. This is known by the detection of Th1cells in the mononuclear cell infiltrate of some progressing tumors.Therefore, the development of a Type 1 adaptive immune response alonemay not be enough to have a curative effect. One of the reasons for thefailure of a Type 1 adaptive immune response to protect is due to potenttumor-mediated immune avoidance mechanisms.

One method of tumor immune avoidance is by tumor-derived suppressivecytokine production. TGF-beta1 production by malignant tumors isessential for tumor progression and is one of the most importantimmunosuppressive cytokines secreted by tumors. Anotherimmunosuppressive tumor-derived cytokine is IL-10. TGF-beta1 and IL-10have been detected in tissue specimens from a variety of tumor types.TGF-beta1, and IL-10 are potent inhibitors of cellular immune functionwhich allows tumors to escape immune surveillance and destruction byCTL.

The Type 1 cytokine storm produced by the method of the presentinvention causes the down regulation of tumor cell production ofTGF-beta1 and IL-10. Therefore, an additional method of the presentinvention comprises introducing a sufficient amount of activatedallogeneic T-cells, preferably CD4+T-cells, into a host in order to downregulate tumor production of immunosuppressive cytokines such asTGF-beta1 and IL-10.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting. Other features and advantages of the inventionwill be apparent from the following detailed description.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

A preferred method for producing allogeneic cells with enhancedproperties for stimulation of the Mirror Effect mechanism of the presentinvention involves: (1) the collection of mononuclear cell sourcematerial by leukapheresis from normal screened donors; (2) the isolationof CD4 T-cells from the source material; (3) the labeling of the CD4+cells with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs); (4) themixing of the labeled CD4+ cells with biodegradable microspheres ornanospheres coated with an agent capable of cross-linking the mAbs onthe CD4+ cells; (5) the concentration of the biodegradable spheres andlabeled CD4+ cells by centrifugation; (6) the culture of the mixture inserum-free media without exogenous cytokines at cell densities in excessof 10⁶ cells/ml; (7) the culture of the cells undisturbed in anincubator for 2 days; (8) the addition of additional labeling agents andcoated biodegradable spheres; (9) centrifugation of the new culturemixture followed by removal of 50-90% of the cell-free culture mediavolume; (10) the passage 90% of the conditioned cell-free culture mediathrough a dialysis filter; (11) bringing the remaining 10% of theconditioned media back to the original volume with fresh culture mediaand adding this replenished conditioned media back to the cell mixture;(12) repeating steps 8 through 11 at least daily for a total cultureperiod of at least 6 days.

Step 1

In practicing the preferred method provided herein, a startingpopulation of mononuclear cells (source material) is collected from adonor, preferably by a leukapheresis procedure. The donors recruited toprovide source material must be healthy and free of adventitious agents.Donors preferably will have completely mis-matched HLA antigens to theintended recipient. While not desired, source material from a partialHLA matched donor (such as a sibling of the intended recipient) can alsobe used in the method of the present invention. Partial matched sourcematerial need only be used if the recipient is so immunocompromised thatinfusion of mis-matched donor cells could cause a GVH disease reaction.Even in the case of immunocompromised individuals, it is stillpreferable to use mis-matched cells. In order to minimize the risk ofGVH disease in these patients, the dose of the mis-matched donor cellscan be reduced or the mis-matched cells could be irradiated just priorto infusion.

Generally, the donors should be carefully screened and such tests foradventitious agents conducted, as would qualify the donor to provideblood for transfusion. Examples of such tests for adventitious agentsshould include, at a minimum, screening for anti-HIV-1, anti-HIV-2,anti-HCV (hepatitis C), anti-HTLV-1 and anti-HTLV-2 antibodies, HbsAg(hepatitis B surface antigen), and syphilis (RPR). In a relatedembodiment, it is also preferable to additionally screen for CMV, and/ormalaria and/or hepatitis G. Blood from any donor that tests positive foradventitious agents should not be used as source material.

Donors generally undergo an 8-12 liter leukapheresis procedure astolerated. Donors do not need to be mobilized. The source material maybe cryopreserved after collection for processing at a later date, butthe material is preferably processed immediately or within 24 hours ofcollection. The leukapheresis source material collected should beprocessed by first being washed to remove plasma proteins,anticoagulant, and to reduce the number of platelets. Suitable washmedia includes PBS (without calcium or magnesium) supplemented with 1-5%human serum albumen (HSA). The washing step can be performed bycentrifuging the cells and removing the supernatant fluid, which is thenreplaced by PBS. The process can be best accomplished using asemi-automated “flow through” centrifuge (COBE 2991 System, Baxter orCytoMate, Baxter). The cells are maintained in a closed system as theyare processed. Washing can be repeated up to 3 times as required.Following the wash, the WBC recovery should be greater than 85% and theplatelet recovery should be less than 40%.

Step 2

The washed source material is next processed to positively select a purepopulation of CD4+ cells. Positive selection is preferred over negativeselection techniques, as positive selection results in a knownend-product and requires less reagents. The preferred method forpositive selection is the use of immunomagnetic technology availablefrom Dynal (Norway) or Miltenyi (Germany). One preferred method topositively select CD4+ cells from the source material is the use ofmagnetic microparticles and the CliniMACS cell separator devicemanufactured by Miltenyi (Germany). The cells are first labeled withanti-CD4-biotin coated monoclonal antibodies and then tagged withanti-biotin magnetic particles (supplied by Miltenyi and used inaccordance with manufacturer's instructions). The solution of labeledcells is then passed over a magnetic filter for retention of the CD4cells.

In order to maintain closed, sterile operations, the labeling of thecells in preparation for CD4 positive selection can be conducted with aCytoMate Cell Washer system (Baxter). This procedure is performed in aclosed sterile disposable kit on the CytoMate device. The CliniMACS CellSeparator then uses a closed sterile disposable kit and a combination ofprograms and reagents to obtain an enriched population of CD4+ cells byperforming an immunomagnetic positive selection on the cells tagged withthe microbeads. The CLiniMACS can process a maximum of 6×10¹⁰ total WBCand 5×10⁹ labeled (CD4+) cells. A leukapheresis protocol normallyresults in the collection of approximately 10¹⁰ mononuclear cells fromwhich approximately 10⁸ purified CD4 cells are normally collected.

Wherever possible during this procedure, a Sterile Connecting Device(Terumo) is used to make a sterile connection between bags and maintaina sterile closed system. Where use of the SCD is not possible,connections are made under strict aseptic conditions in a Laminar FlowBiosafety Cabinet.

In the positive selection of CD4+ cells, it is most important toeliminate CD8+ cells from the source material, as contaminating CD8+cells can outgrow the CD4+ cells in subsequent steps in the process ofthe invention. Macrophage contamination is common after CD4+ cellpositive selection. This may be due to the fact that some macrophagepopulations express the CD4 molecule. However, macrophages will die outin subsequent steps in the process and are not normally a great concern.Similarly, B cells will also not live through subsequent processingsteps. In rare instances, macrophage contamination will cause CD4+ celllysis or inhibition of CD4+ cell proliferation. In these cases, amacrophage reduction step prior to CD4+ cell selection might beindicated. Macrophage reduction can be accomplished by a variety ofmethods recognized in the art, including pre-incubation on plastic,passing through nylon wool columns or through ingestion of magneticbeads and subsequent removal in a magnetic field.

The purified CD4 cells will be mostly naïve cells with a phenotype ofCD4+, CD45RA+, CD62L^(Hi). Contamination with up to 40% memory cellswith a phenotype of CD4+, CD45RO+, CD62L^(lo) will not affect theprocess. However, if memory cells are in excess of 40% at this step inthe process, this usually indicates that the donor is not normal andthus the batch should be rejected and not used to develop cells forinfusion. The purified CD4 cells can be stored at room temperature forup to 24 hours.

Step 3

The next step in the process is the ex-vivo culture of the purified CD4+cells. It is preferred that the CD4 cells be exposed to a persistent andconstant activation stimulus for at least 6 days. In order to activatethe cells, they are first preferably labeled with activating agents,such as anti-CD3 and anti-CD28 mAbs and the activating agents are thencross-linked to deliver an activation signal to the CD4 cells. To labelthe cells, the cells are first adjusted to a cell density of 10⁷ cellsper ml in serum-free culture media. A normal batch would contain around10⁸ CD4 cells in 10 ml of media. The mAbs are each added to the cells ata final concentrations of at least 1 microgram per ml, preferably 10micrograms per ml. The cells should be incubated with the mAbs on arotating or end to end mixing device for 15 to 30 minutes at roomtemperature or preferably at 4° C. The cells should then be washed toremove excess mAbs and resuspended at 10⁷ cells per ml in serum-freeculture media.

Step 4

The preferred cross-linking method is to mix the labeled cells withbiodegradable nanospheres or microspheres coated with an agent reactiveto the activating agents. For example, the biodegradable spheres can becoated with a mAb specific for the Fc region of the anti-CD3 andanti-CD28 mAbs, or in the case where the activating agents are mousederived, the coating agent could be a polyclonal anti-mouse antibody.The labeled cells are mixed with the coated biodegradable microspheresat a sphere to cell ratio of at least 1:1, preferably a minimum of 3:1,and most preferably a minimum of 5:1. The sphere/cell mixture ispreferably mixed well with the labeled cells for 15 to 30 minutes atroom temperature, or preferably at 4° C.

Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolicacid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone)(PCL), and polyanhydrides are preferred materials for use asbiodegradable polymers for the nanospheres/microspheres. Thebiodegradable composition should be designed to degrade in physiologicalmedia within 7 days, more preferably within 3 days.

In a preferred embodiment of the present invention, the biodegradablespheres are constructed from a linear polyester polymer containing amixture of lactic acid and glycolic acid. This class of polymers meetsthe requirements of biocompatibility and biodegradation into harmlessend products for use in human biological drug preparations. Thesepolymers, hereinafter referred to as PLGA, are degraded by esterhydrolysis into lactic acid and glycolic acid which are metabolized inthe body into carbon dioxide and water. PLGA has been shown to possessexcellent biocompatibility. The innocuous nature of PLGA can beexemplified by the approval by the regulatory authorities, including theU.S. Food and Drug Administration, of several parenteral delayed releasepreparations based on these polymers.

Copolymers of DL-lactate and glycolide, rather than L-lactate andglycolide, are preferred because they are amorphous when DL-lactate is amajor component, as opposed to semicrystalline when L-lactate is a majorcomponent. This property decreases the degradation time of the polymer.The inherent viscosity (abbreviated as “I.V.”; units are indeciliters/gram) of the polymer is a measure of its molecular weight.Preferably, the inherent viscosity of the polymer is from about 0.10dL/g to about 1.0 dL/g (as measured in chloroform), more preferably fromabout 0.10 dL/g to about 0.50 dL/g and most preferably from 0.10 to 0.30dL/g.

Suitable biodegradable polymer material is a 50/50 mixture ofpoly(DL-lactide co-glycolide). The polymer can be purchased fromcommercial suppliers such as Birmingham Polymers, Inc (Birmingham, Ala.)under the trade name Lactel®. The 50/50 DL-PLG product number 50DG020with a inherent viscosity of 0.15 to 0.25 dl/g is a preferred materialfor use in the present invention. Another preferred material is 50/50DL-PLG with an inherent viscosity of 0.32 to 0.44 dl/g manufactured byBoehringer Ingelheim (Ingelheim, Germany) under the trade name Resomer®RG 503. Another preferred material is Lactel® 50/50 DL-PLG productnumber 50D040 (Birmingham Polymers) with a 0.26 to 0.54 inherentviscosity.

Microspheres or nanospheres can be prepared by various known methods,including solvent evaporation, phase separation, spray-drying, orsolvent extraction at low temperature. The process selected should besimple, reproducible and scalable. The resulting microspheres should befree-flowing and not aggregates in order to produce a uniformsyringeable suspension. The microspheres must also be sterile. This canbe ensured by a terminal sterilization step and/or through asepticprocessing.

In a preferred embodiment, the solvent evaporation method is utilized toproduce the spheres. To produce microspheres or nanospheres with thismethod, the hydrophobic 50/50 DL-PLG polymer is dissolved in awater-immiscible organic solvent to give a polymer solution. Thesolution is then added into an aqueous solution of a surfactant to forman emulsion system and stirred. The faster the stirring speed, thesmaller the size of the microspheres. Micro spheres are obtained bysubsequently evaporating the solvent by continuous stirring, which canbe under vacuum or heat.

The water-miscible organic solvents need to be non-toxic to the body.Typical examples of organic solvents are members selected from the groupconsisting of acetic acid, lactic acid, formic acid, acetone,acetonitrile, dimethyl formamide, dimethyl acetamide, dimethylsulfoxide, dioxane, and N-methyl pyrrolidone and mixtures thereof.Preferably, the water-miscible organic solvent is a member selected fromthe group consisting of acetic acid, lactic acid, N-methylpyrrolidone,or a mixture thereof. The water-miscible organic solvent may be usedalone or in a mixture with water.

The aqueous phase can contain an emulsion stabilizer that is preferablysoluble in water and alcohol, is capable of increasing viscosity of thesuspending medium (water-miscible alcohol) when dissolved in the medium,is non-toxic to the body and causes no environmental problems. Typicalexamples of emulsion stabilizer solutions are: water-soluble syntheticpolymers such as polyvinylpyrrolidone, poly(ethylene glycol), andpoloxamer; cellulose derivatives such as hydroxypropyl cellulose andhydroxypropylmethyl cellulose, and preferably, polyvinylpyrrolidone andhydroxypropyl cellulose. The content of emulsion stabilizer in thewater-miscible alcohol is preferably within the range of 0.1.toabout.50% (w/v), and more preferably within the range of 0.2.to about20% (w/v). The content of emulsion stabilizer can be varied according tothe viscosity of the water-miscible alcohol needed.

The water-miscible alcohol, wherein the emulsion stabilizer isdissolved, is stirred at a temperature of 10 about. 80 degrees C.,preferably from 20.about.60.degree. C., and most preferably at roomtemperature at a speed of 200.to about. 20,000 rpm, preferably at aspeed of 800 to 2000 rpm. The polymer solution is slowly added to thewater-miscible alcohol wherein the emulsion stabilizer is dissolved, andthe mixture is stirred from 5 minutes to about. 60 minutes. Stirring canbe continued for up to 5 hours to allow evaporation of the organicsolvent. The resulting microspheres can then collected by centrifugationand washed extensively. The washed microspheres are then ready forattachment of the cross-linking material.

The diameter of the microspheres prepared should preferably be withinthe range from 0.01 to 300 um, and more preferably within the range from0.1 to 100 um. and most preferably between 0.1 and 10 um. The particlesize (diameter of the microspheres) can be controlled by adjusting thestirring speed during processing, the viscosity of the water-misciblealcohol, and the viscosity of the polymer solution.

Post-coating of the biodegradable spheres with the cross-linkingmaterial can be accomplished by a variety of standard methods. Inpreferred embodiments, first materials that are proteins can be bond tothe biodegradable microspheres by adsorption with standard knownmethods. A preferred method for adsorbing a protein to the biodegradablespheres is to suspend the microspheres in 0.1M Borate buffer at pH 8.5,spin down and resuspend the microspheres 2 or 3 times. The cross-linkingprotein, for example goat anti-mouse polyclonal antibody, is thensuspended in the borate buffer at a concentration of 10 micrograms/mland added to the microspheres at a density of 2×10⁸ spheres per ml. Themixture is mixed end-to-end for at least 4 hours and for up to 24 hours.The mixing is preferably conducted at 4° C. After mixing, themicrospheres are spun and the supernatant removed and analyzed forprotein determination. The coated microspheres are then resuspended in aphysiological buffer, such as phosphate buffered saline containing ablocking agent, such as 1-5% bovine or human serum albumen and/or 0.05%w/v Tween 20.

Step 5

In order to enhance the activation signals to the CD4 cells, the wellmixed labeled cell/sphere mixture is spun down in a centrifuge at 500 to800 rpm at 4° C. for 2 to 10 minutes. The force should not be so greatas to tightly “pellet” the cells, but just great enough to concentratethe cells. The centrifugation forces the cells and the spheres tointeract, increasing the cross-linking and the signal transduction tothe CD4 cells, providing enhanced activation. The cells are preferablyspun while in the gas permeable bag culture container. Aftercentrifugation, the cells are gently resuspended by massage andagitation of the flexible bag container and placed in an incubator in anatmosphere of 5% carbon dioxide at 37° C.

Step 6

It is also preferable that the CD4 cells be kept in close cell-to-cellcontact during the ex-vivo culture process. Close cell-to-cell contactcan be accomplished by culturing the cells at a high cell density,preferably at 10⁶ cells per ml or greater. It is also desirable tosubject the cells to frequent centrifugation in order to enhancecell-to-cell contact and the delivery of activation signals.

The purified and labeled CD4+ cells mixed with the coated biodegradablespheres should initially be suspended in culture media at a cell densityof 10⁶ cells per ml and at a sphere to cell ratio of not less than 1:1,preferably greater than 3:1, and most preferably greater than 5:1.X-VIVO15 (BioWhittaker) is a preferred culture media. If the cells tendto stick to the culture containers, the culture media can besupplemented with 1% human serum albumen (HSA). The preferred culturecontainers are gas permeable plastic bags, such as LifeCell (BaxterOncology, Dearfield, Ill.).

Step 7

For the first 2 days of culture, the cells should be left undisturbed inthe incubator.

Step 8

On the third day, additional microspheres and mAbs are added to theculture and mixed thoroughly. To a 100 ml culture, 100 micrograms eachof anti-CD3 and anti-CD28 mAbs are added together with 3-5×10⁸ coatedbiodegradable microspheres.

Step 9

Maintaining cells at high densities in culture with biodegradablespheres requires the frequent changing of the culture media. The highcell densities result in a high rate of build up of metabolic wasteproducts and consumption of available nutrients. In addition, thehydrolysis of the biodegradable spheres causes the pH of the culturemedia to become acidic. Too rapid media replacement, however, can bedetrimental to cultures where exogenous cytokines are not utilized. Itis preferable not to use exogenous cytokines when processing cells foruse in cell therapy protocols, as exogenous cytokines can be toxic wheninfused into humans and can make the cultured cells dependant upon thepresence of the exogenous cytokines for viability. Therefore, themethods of the present invention include a dialysis step in the cellprocessing.

In order to remove 50-90% of the media and to enhance the activationstate of the cultured cells, the fresh mixture of mAbs and spheres isagain spun in a centrifuge as in step 5 in order to concentrate thecells enough to remove cell-free supernatant. This process can berepeated several times a day if required in order to keep the pH of theculture between 7.0 and 7.2.

Step 10

Dialysis of the removed culture medium through a membrane with a poresize of 10,000 Daltons or less will enable retention of endogenouscytokines while allowing passage of metabolic waste. In preferredembodiments, 50-90% of the culture medium of a culture is removed atleast daily and 90% of the removed media passed through a dialysisfilter.

Step 11

The media passed through the dialysis filter is discarded, while the 10%retained media is brought up to the original volume with fresh culturemedia and then added back to the T-cell/sphere culture. The retainedmedia will contain the endogenous cytokines at the same concentrationsas before the removal of the culture media.

Step 12

Steps 8 through 11 are repeated at least once a day for a minimum of 3days (6 days total in culture). In a typical batch run of the process,the cultures are initiated with approximately 10⁸ purified CD4 cells in100 ml of culture media volume (day 1). By the method described, thecells will expand to approximately 1-5×10⁹ cells by day 6 to day 8. Uponreaching this cell number, the cells can be resuspended in 1000 ml ofculture media in a gas permeable bag and steps 8 through 11 repeated atleast daily for up to an additional 3 to 6 days (day 9 to day 14 ofculture). Over this time, the total cells in the culture will expand toapproximately 1-5×10¹⁰ cells.

Harvest

The cells can be harvested any time after day 6 of culture or when atleast 10⁹ cells are available in the batch culture. To assure maximalcytokine production, the timing of the harvest should occur such thatthe cells are formulated and infused 24 hours after the last step 8-11cycle.

The cells produced by the methods of the invention can be aliquoted intomultiple dosages of at least 10⁸ cells, preferably at least 10⁹ cells.The aliquoted dosages of cells can be frozen for storage prior toinfusion. In the case of a frozen dosage form, the cells are frozen incryoprotective media supplemented with conditioned media from thepreparatory cell culture in order to maintain high cell viability.Frozen dosages are thawed, activated and formulated within 24 hours ofinfusion.

Formulation

The harvested cells are formulated with the activating mAbs attached tothe cells surface being cross-linked with the coated biodegradablemicrospheres, in order to assure the cells remain activated at the timeof infusion and while in circulation.

The mixture of CD4 cells and microspheres are suspended in infusionmedium (e.g., isotonic solutions such as normal saline, 5% dextrose,Plasma-Lyte (Baxter) or Normasol (Abbott)). In some embodiments, theinfusion medium is supplemented with 0.5%-10% human serum albumen (HSA).

The mixture is preferably adjusted to a final T-cell concentration ofbetween 1×10⁷ to 1×10⁸ cells per ml of infusion media. In a preferredembodiment, 10⁹ T-cells are formulated in 100 ml of infusion media. Theformulation is then packaged in one or more containers, such assyringes, plastic pouches, or plastic bottles.

Infusion

A sufficient number of formulated CD4 cells are administered to therecipient in order to ameliorate the symptoms of the disease. Typically,dosages of 10⁷ to 10¹⁰ cells are infused in a single setting, preferablydosages of 10⁹ cells. Infusions are administered either as a single 10⁹cell dose or preferably divided into several 10⁹ cell dosages. Thefrequency of infusions can be every 3 to 30 days or even longerintervals if desired or indicated. The quantity of infusions isgenerally at least 1 infusion per patient and preferably at least 3infusions, as tolerated, or until the disease symptoms have beenameliorated. The cells can be infused intravenously at a rate of 50-250ml/hr.

It is important that the infused cells express high levels of FasL andCD40L. In addition to IFN-gamma, the cells should also produce thefollowing Type 1 cytokines: IL-2, IL-15, TNF-alpha and TNF-beta. Thecells should not express CTLA-4 on their surface and should not produceTGF-beta, IL-4 or IL-10. Upon co-culture with allogeneic peripheralblood mononuclear cells, the cells should cause the upregulation of Type1 cytokines IL-1, IL-12, TNF-alpha and IFN-gamma and upregulation of MHCand co-stimulatory molecules on autologous APC and target cells. Inaddition, upregulation of effector molecules such as FasL, TRAIL, TWEAKand other TNFR should be evident in autologous cells after mixing withthe allogeneic CD4 cells produced by the method of this invention:

Mechanism of Action

Cells resulting from the method of the invention will acutely activatecells of the innate immune system when co-cultured. This activationoccurs due to interaction with CD40L expressed on the cells produced bythe method of the invention and the CD40 molecule expressed on hostinnate immune cells. Upon co-culture of host PBMC and allogeneic donorcells produced by the method of the invention, macrophages and dendriticcells upregulate co-stimulatory cell surface molecules and MHC class Iand II molecules, produce pro-inflammatory cytokines, such as IFN-gamma,TNF-alpha, IL-1, IL-12 and Type I interferons. This creates a “cytokinestorm” that is nearly identical to the cytokine storm environmentcreated by infusion of allogeneic donor lymphocytes in BMT protocols.

These characteristics combined with the ability of the activated hostmacrophages and dendritic cells to uptake (by phagocytosis andendocytosis) and subsequently destroy tumor cells and pathogenicorganisms enables the enhanced presentation of the antigenic products ofthese pathogens and tumors via the MHC class I and II pathways toantigen reactive T-cells. Further, the surface phenotype of the cellsproduced by the method of the invention (CD45RO+, CD44+, CD62L^(lo))will enable the infused cells to traffic to sites of inflammation anddeliver their Type 1 cytokines to the microenvironment. This cansuppress local Type 2 cytokine production, upregulate MHC Class I and IIexpression, co-stimulatory molecule expression and recruit tumoricidalmacrophages to the tumor bed.

The high expression of FasL and TRAIL on the cells produced by themethod of the invention, combined with effector activity of innateimmune cells recruited to the site of inflammation or tumor bed willcause apoptosis and antigen shedding to the draining lymph nodes. Thelymph nodes should be populated with activated dendritic cells from theinitial CD40L/CD40 interactions and be primed to present antigens to theadaptive immune system components in a cytokine environment favorable toType 1 immune response development. CD40L/CD40 activation of dendriticcells causes production of IL-12 and TNF-alpha by dendritic cells,cytokines which are known to bias activated naïve T-cells to Th1 andType 1 adaptive immunity. Further, IL-12 production will further induceIFN-gamma production from T-cells and NK cells which will in turnfurther upregulate IL-12 from macrophages, creating an autocrinefeedback loop which drives macrophage activation, T-cell maturation toType 1 immunity and amplifies innate NK activity.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Methods:

Microsphere Preparation

The solvent evaporation method was used for preparation of microspheres.Lactel® (Birmingham Polymers, Birmingham, Ala.) 50/50 DL-PLG productnumber 50DG020 with a inherent viscosity of 0.15 to 0.25 dl/g was usedas the polymer. The DL-PLG powder was dissolved in 20 ml of methylenechloride to a final 5% DL-PLG w/v ratio. The 5% DL-PLG solution was thenadded dropwise to 1251111 of 2.4% hydroxypropylmethylcellulose in 0.1Mglycine/HCl buffer pH 1.1 under constant stirring at 1000 rpm at roomtemperature (25±2° C.). Stirring was maintained until completeevaporation of the organic solvent (about 3 hours). Microspheres werecollected by centrifugation at 1000 rpm, 5 min at 40 C followed by threecycles of washing with distilled water, filtered and dried overnight.The microsphere sizes ranged from 3.0 to 7.0 um with a CV maximum of<10%. The spheres were then coated with polyclonal goat anti-mouseantibody using the absorption method. The antibody was suspended in 30ml of PBS solution with 5% human serum albumen (HSA) at a concentrationof 10 ug/ml. This solution was used to resuspend the dried microspheresat a concentration of approximately 2×108 particles per mi. Themicrospheres and the polyclonal antibody were mixed end over end at 40 Cfor 8 hours. The microspheres were then washed 3 times in PBS with HSA,filtered and dried. The dried particles were stored in a solution of 70%isopropanol prior to use.

Allogeneic Cell Product Preparation

For the examples below, Allogeneic Cell Product was prepared accordingto the method described in the preferred embodiments. Briefly, 1.2×10¹⁰peripheral blood mononuclear cells (PBMC) were collected from a healthydonor by leukapheresis. The PBMC were washed and stored a roomtemperature overnight. The PBMC were enriched for CD4+ cells by labelingwith biotinylated anti-CD4 mAb and mixing with a secondary anti-biotinmAb magnetic particles (Miltenyi Biotec, Germany). The CD4+ cells werethen selected by passing through a magnetized column (MACK)). 1.3×108CD4+ were selected and placed in 100 ml of XVIVO-15 culture media in aLifeflask (Baxter) gas permeable bag. The CD4+ cells were incubatedovernight at 370 C in an atmosphere of 5% CO2. The following day, thenon-adherent cells were washed and labeled with anti-CD3 and anti-CD28mAbs and suspended with goat anti-mouse coated biodegradablemicrospheres at a 3:1 ratio. The suspension was centrifuged at 1000 rpmfor 5 min and gently resuspended by manual massage of the culture bag.The suspension was incubated for 72 h, and the cells were relabeled andsuspended with new microspheres. The suspension was centrifuged at 1600rpm for 8 min, the supernatant removed and 90% of the volume passedthrough a dialysis filter. The retained supernatant was added back tothe cell suspension and the volume brought back to 100 ml with freshculture media. This process was repeated daily until day 9 of culture.On day 10, the resulting cells were used in the examples describedbelow.

Example #1 Phenotypic Analysis of Allogeneic Cell Product

A sample of allogeneic cell product was taken on day 1 and day 10 forphenotypic analysis. For cell immunophenotyping, surface labeling wasperformed by a direct fluorescence technique using monoclonal antibodies(Becton-Dickinson, San Jose, Calif., USA), against human CD4, CD8, CD14,CD19, CD56, CD4/CD25, CD4/DR, CD4/CD45RA, CD4/CD45RO, CD4/CD62L,CD4/CD154 (FasL), CD4/TRAIL. To detect intracellular cytokines,mononuclear cells were permeabilized with FACS permeabilizing solution(Becton-Dickinson). Flow cytometry analyses were carried out with aFACSort equipment (Becton-Dickinson) using the Cellquest software. Theresults are reported as the percent of stained cells calculated from10,000 events for all immunophenotypes.

Results in Percentage of Total Cells (MFIR):

DAY 1 DAY 10 CD4 92.5 99.8 CD8 0.8 0 CD14 4.8 0 CD19 0.9 0 CD56 1.7 0CD4/CD25 2.3 92.9 CD4/DR 4.5 89.7 CD4/CD45RA 70.3 10.9 CD4/CD45RO 16.678.1 CD4/CD62L^(hi) 69.4 0.9 CD4/CD154 (FasL) 0.8 74.3 (67)   CD4/TRAIL0.3 68.3 (26.6) CD4/IFN-gamma 18.6 98 CD4/IL-4 4.8 0.2

These results indicate that the Allogeneic Cell Product hasdifferentiated into a Type 1 cell with an activated memory phenotype.

Example #2 Cytokine Gene Profile of Allogeneic Cell Product

To determine the cytokine profile of the Allogeneic Cell Product,cytosolic RNA was purified using a RNeasy kit (Qiagen) and reversedtranscribed using a Roche First Strand cDNA synthesis kit. Primers andprobes were purchased from Applied Biosystems or were designed usingPrimer Express software. Real-time PCR amplification and productdetection was performed according to manufacturer's recommendedprocedures on an ABI Prism 7700. Gene product is expressed relative toGAPDH expression, which is set at a value of 100,000 on day 1 and day10.

Day 1 Day 10 IL-1beta 85 7 IL-2 4 18,450 IL-4 2 0 IL-5 0 0 IL-6 0 0IL-10 11 10 IL-12p35 12 12 IL-12p40 0 0 IL-13 82 3 IL-15 11 1200 IL-1810 8 TNF-alpha 21 84,880 IFN-gamma 18 94,600 TGF-beta 0 0

Example #3 Host PBMC Rejection of Allogeneic Cell Product

PBMC from a stage 3 ovarian cancer patient was prepared by densitygradient purification and isolation of buffy coat. The host PBMC weremixed with Allogeneic Cell Product at a 50:50 ratio and cultured in 24well plates for 7 days. The Allogeneic cells were labeled with greencell tracker dye, 5-chloro-methyl-fluorescein diacetate (CMFDA). Thecultures were set up in triplicate.

Results:

At the end of the 7 day culture, less than 2% of the live cells in eachof the wells stained green, indicating that they were rejected by thehost PBMC.

Example #4 Cytokine Analysis of mixed Host PBMC and Allogeneic Product

In order to determine the ability of the allogeneic cell productproduced by the method of the invention to stimulate host cancer patientPBMC to produce Type 1 cytokines, allogeneic cells were prepared asdescribed in the Preferred Embodiments, harvested on day 9 and mixedwith 1×10⁶ PBMC from a cancer patient in a 24 well culture plate andincubated for 48 hours at 37° C. in a humidified atmosphere containing5% CO2.

Human PBMC were isolated by density gradient centrifugation ofperipheral blood obtained from a patient with metastatic breast cancerprior to mastectomy. Allogeneic cell product was added to the PBMCcultures at ratios of 1:100, 1:50 and 1:25. PBMC in media alone was usedas a negative control and PBMC activated with PHA served as the positivecontrol.

After 48 hours, supernatant samples were removed from each well andanalyzed by ELISA. Results are shown as means+/−SE of triplicatecultures in pg/ml. ND=not detectable.

Results:

Media PHA 1:100 1:50 1:25 IL-2 83 ± 8 12934 ± 24  18734 ± 73  16726 ± 8212993 ± 72  IL-4 249 ± 2   643 ± 12  32 ± 3 ND ND IL-6 349 ± 12 1034 ±18 1395 ± 15 1863 ± 1 1822 ± 18 IL-10 874 ± 32 1739 ± 52 ND ND NDIL-12p70 ND 980 ± 6 3890 ± 54  4176 ± 32 4231 ± 31 IL-15 ND 1628 ± 482847 ± 91  7493 ± 93 8328 ± 74 IFN-alpha 42 ± 3 349 ± 7  843 ± 34  938 ±23 1022 ± 34 IFN-gamma ND 380 ± 5 15863 ± 532 178745 ± 368 22903 ± 839TNF-alpha ND 1893 ± 32 11932 ± 323  12435 ± 393 13458 ± 239

The results indicate that the allogeneic cell product of the presentinvention can elicit strong upregulation of Type 1 cytokine productionand down regulate Type 2 cytokine production.

Example #5 Phenotypic Analysis of Host Cells After Mixed with AllogeneicProduct

Host CD3+ T-cells and CD14+ monocytes from Example #3 were analyzedphenotypically for effector and co-stimulatory markers.

Results in Percent of Total PBMC (MFIR)

Day 1 Day 7 CD14/CD80 12.9 66.5 (5.8) CD14/CD86 16.6 81.7 (922)CD3/CD154 (FasL) 0.6 34.3 (47) CD3/TRAIL 0.2 38.5 (16.6) CD14/CD154 11.635.3 (13.8) CD14/TRAIL 4.8 28.4 (9.5)

The results indicate that host cells upregulated co-stimulatory andeffector molecules during the rejection of the Allogeneic Cell Productand in the presence of the Cytokine Storm.

Example #6 Stimulation of NK Cytotoxicity

NK activity against K562 target cells was assessed by a flow cytometryassay using the DIO membrane dye (Molecular Probes, Eugene, Oreg., USA)to stain live K562 cells and propidium iodide (Sigma) nuclear dye tostain dead cells. The percent of specific lysis was calculated by theformula:

$\frac{\%\mspace{14mu}{dead}\mspace{14mu}{target}\mspace{14mu}{cells}}{100 - {\%\mspace{14mu}\left( {{debris}\mspace{14mu}{and}\mspace{14mu}{fragments}} \right)}} \times 100$

PBMC from a cancer patient were incubated in media alone and supernatantfrom a 48 h co-culture of the allogeneic product and autologous PBMC ata 1:100 ratio.

Results:

Effector:Target Ratio 100 50 25 12.5 6.3 3.1 media 8.34 4.32 2.54 1.080.86 0.34 supernatant 85.52 82.11 71.23 50.65 34.55 20.91

These results indicate that the Type 1 cytokine storm elicited by themethod of the invention is capable of significantly enhancing host NKactivity.

Example #7 Cytokine Storm Supernatant Effects on Tumor Immunogenicity

Cancer cell lines NCI-H23 (lung cancer), Caki-1 (renal cell cancer) andACHN (renal cell cancer) were analyzed for expression of MHCI, MHCII,death receptors Fas and TRAIL-R2 and co-stimulatory molecules CD80 andCD86. The cell lines were then cultured in the bottom of a transwellplate. In the top well host PBMC from a normal donor and the AllogeneicCell Product were mixed at a 100:1 cell ratio. The cultures wereincubated for 96 hours.

Results:

Results in MFIR Day 0 Day 4 NCI-H23 MHCI 220 780 MHCII 0.8 6.8 CD80 0.44.8 CD86 280 550 Fas 0.8 18.5 TRAIL-R2 19.9 20.8 ACHN MHCI 190 1387MHCII 0.2 8.8 CD80 0.4 3.8 CD86 180 988 Fas 0.7 28.5 TRAIL-R2 7.9 10.8Caki-1 MHCI 120 569 MHCII 0.8 6.8 CD80 0.4 4.8 CD86 150 650 Fas 0.8 18.5TRAIL-R2 1.9 20.8

These results indicate that the cytokine storm elicited by the method ofthe invention is capable of increasing the immunogenicity of tumor cellsand their susceptibility to apoptosis.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A method of treating a diseased host comprising:stimulating the host's Type I adaptive immunity and generating de novotumor associated antigen (TAA) shedding in the host wherein thestimulating is performed by the administration of activated allogeneicCD4+ cells.
 2. The method of claim 1 wherein the CD4+ cells comprisecross-linked CD3 and CD28 surface molecules.
 3. The method of claim 1wherein the method further comprises activation and upregulation ofcytotoxic activity of the host's NK cells.
 4. The method of claim 1wherein the TAA shedding is by apoptosis of the tumors.
 5. The method ofclaim 4 wherein apoptosis is induced by binding of Fas with FasL.
 6. Themethod of claim 4 wherein apoptosis is induced by TRAIL ligand andTRAIL-R.